Gram-negative bacillary infections have become increasingly important causes of morbidity and mortality during the past 30 years. Evaluation of pathophysiologic events during such infections have demonstrated that the major cell wall component of Gram-negative bacteria, endotoxin (LPS), induces an extensive variety of biologic activities in almost all mammalian species. The multiple effects of LPS have directed attention from potential effects of other cell wall components of Gram-negative bacteria. Recent studies of proteins, termed endotoxin associated protein (EAP), which are closely associated with LPS have demonstrated a variety of biologic activities similar to, but distinct from those of LPS. Others have demonstrated that EAP was mitogenic for B lymphocytes from endotoxin-resistant C3H/HeJ mice and human lymphocytes and induced expression of B lymphocyte surface antigens. Microphage effector activities, increased tumorcidal activity, glucose utilization, and enzyme secretion, have also been shown to be inducible by EAP. Studies in our laboratory have shown that EAP is a potent inducer of the acute phase reactant, serum amyloid A (SAA), in endotoxin resistant mice. EAP appeared to act directly on hepatocytes to induce SAA in contrast to LPS whose SAA inducing activity is mediated through production of a macrophage effector. EAP also appeared to have Interleukin-1 (IL-1) like activity and also to induce ll-1 production by macrophages from endotoxin resistant mice in the LAF assay. EAP also exhibited potent Granulocyte Colony Stimulating activity with production of eosinophile colonies from human peripheral blood depleted of monocytes and lymphocytes. EAP also induced maturation of cells of the undifferentiated human leukemic cell line, HL 60. The crude EAP preparation studied demonstrated 9 bands on PAGE. Active material was found in the void volume after chromatography on Sephadex G75 and demonstrated the presence of only 4 bands of 29,800, 26,900, 22,600 and 20,200 daltons on PAGE. Heating to 90 degrees Cx45 min., trypsin and pronase treatment ailed to destroy biologic activity. The present studies are intended to further separate and isolate the individual components of EAP utilizing column chromatography, high pressure liquid chromatography and isoelectric focusing. Separation procedures will be monitored for biologic activity using the SAA assay and lymphocyte mitogenicity with C3H/HeJ mice and colony stimulating activity on peripheral and bone marrow cells. Mechanisms involved in the direct colony stimulating activity and differentiation of the HL 60 leukemic cell line will be examined. The role of EAP on other biologic activities tributed to LPS, especially those mediated by monocyte effectors also will be examined.